Many cancers are believed to result from a series of genetic alterations leading to progressive disordering of normal cellular growth mechanisms (Nowell, Science 194:23 (1976), Foulds, J. Chronic Dis. 8:2 (1958)). In particular, the deletion or multiplication of copies of whole chromosomes or chromosomal segments, and higher level amplifications of specific regions of the genome are common. See, for example, Smith, et al., Breast Cancer Res. Treat., 18: Suppl. 1: 5-14 (1991), van de Vijer & Nusse, Biochim. Biophys. Acta. 1072: 33-50 (1991), Sato, et al., Cancer. Res., 50: 7184-7189 (1990). In fact, the amplification and deletion of DNA sequences containing proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumorigenesis. Dutrillaux, et al., Cancer Genet. Cytogenet., 49: 203-217 (1990).
In particular, losses and gains of genetic material have been associated with glioma progression (Mikkelson et al. J. Cellular Biochm. 46:3-8 (1991)). In particular, it is believed gains and losses of genetic material serve as signposts for oncogenes and tumor suppressor genes in gliomas. Although gliomas are the most common primary neoplasms of the central nervous system, only modest progress has been made in diagnosing and treating malignant gliomas (Silverberg et al. CA Cancer J. Clin. 40: 9-26 (1990)). The identification of the genetic events leading to neoplastic transformation and subsequent progression aid in elucidating the biological basis for disease, permit early tumor detection and prediction of therapeutic response thereby improving the disease prognosis.
Cytogenetics is the traditional method for detecting amplified or deleted chromosomal regions. The resolution of cytogenetic techniques is limited, however, to regions larger than approximately 10 Mb (approximately the width of a band in Giemsa-stained chromosomes) because of the complex packing of DNA into the chromosomes. In complex karyotypes with multiple translocations and other genetic changes, traditional cytogenetic analysis is of little utility because karyotype information is lacking or cannot be interpreted. Teyssier, Cancer Genet. Cytogenet., 37: 103 (1989). Furthermore conventional cytogenetic banding analysis is time consuming, labor intensive, and frequently difficult or impossible.
Recent methods assessing the amount of a given DNA sequence in a chromosome are more sensitive than the traditional cytogenetic techniques. These methods employ cloned DNA or RNA probes that are hybridized to chromosomal DNA. This method is effective even if the genome is heavily rearranged so as to eliminate useful karyotype information. However, this Southern blotting method gives only a rough estimate of the copy number of a DNA sequence, and does not provide any information about the localization of that sequence within the chromosome.
Comparative genomic hybridization (CGH) is a more recent approach to identify the presence and localization of amplified or deleted sequences (See, e.g., Kallioniemi et al. Science 258: 818-821 (1992)). CGH, like Southern blotting, reveals amplifications and deletions irrespective of genome rearrangement. It produces a map of relative DNA copy number as a function of chromosomal location by comparing the hybridization efficiency of tumor and reference DNA to metaphase chromosomes. CGH, however, has advantages over Southern blotting, providing a more quantitative estimate of copy number, and also providing the location of amplified or deleted sequences in the normal chromosome. Where a deletion or amplification is limited to the loss or gain of one copy of a sequence, the CGH resolution is usually about 5-10 Mb.
Unfortunately, the sensitivity of both CGH and Southern blotting techniques to contamination of tumor samples by normal cells makes estimating the copy number of particular chromosomal sequences within the tumor cell population very difficult. In addition, another problem with conventional CGH is the presence of artifacts due to differential binding of digoxigenin- and biotin-labeled probes at specific chromosomal regions.